Under almost all conditions using any method, the levels of gene transfer to any cell are low because many barriers exist for the efficient delivery of genes to cells. Taken one step further, gene transfer to tissues within living animals is even worse, at least in part due to these and additional barriers that arise from the architecture of th tissue, mechanical forces within these tissues, and the host's response to exogenous materials. The primary goal of our laboratory is to identify and overcome the intracellular barriers to promote effective gene transfer both in vitro and in vivo. Exogenous DNA, either viral or non-viral, must cross the plasma membrane into the cell, travel through the cytoplasm and the cytoskeletal networks, cross the nuclear envelope, localize to specific regions within the nucleus, and be transcribed in order for gene therapy to be successful. In 2003, we demonstrated that when multiple cell types were exposed to mild equibiaxial stretch, their ability to take up and express foreign DNA was 10-fold more efficient than cells grown under static conditions. We have since shown that such stretch reorganizes the cytoskeleton and concomitantly increases the numbers of stable, acetylated microtubules by inhibiting HDAC6, the major cytoplasmic a-tubulin deacetylase. We exploited this information to improve intracellular DNA movement and transfection efficiency by using pharmacologic inhibitors of HDAC6 in cells and animals. More recently we have focused on how plasmids move along modified and unmodified microtubules for their trafficking to the nucleus during transfection and gene transfer and have identified the constituents of the protein-DNA complexes that form immediately after entry into the cytoplasm and at various times afterward using mass spectrometry and proteomics. Further, by comparing the protein complexes on plasmids that productively traffic through the cell with those on plasmids that do not move, we have been able to identify key proteins that control DNA movement and we hypothesize that modulation of these proteins may be used to further improve gene delivery. Finally, we are also looking at how plasmids traffic once inside the nucleus and have found that plasmids show highly dynamic intranuclear movement that can be used to control gene expression. Plasmids localize to distinct regions within the nucleus based on their sequences and we hypothesize that we can control this movement to optimize gene expression. The experiments in this competitive renewal will dissect pathways used for intracellular trafficking of proteins and DNA-protein complexes in both the cytoplasm and nucleus to enhance gene delivery. The specific aims are to (1) Determine the role of tubulin acetylation in cytoplasmic trafficking of transfected plasmids; (2) Identify and characterize the composition of the active DNA trafficking complex; and (3) Determine how plasmids move within the nucleus and how this regulates gene expression.